Importantly, both the miRNAs showed association with disease severity as miR-133b was downregulated by 8.45 fold in acute coronary syndrome (ACS), 3.38 fold in Stable angina (SA) and 2.08 fold in NCA. MiR-21 was upregulated by 2.46 fold in ACS, 1.90 fold in SA and 1.12 fold in NCA. Moreover, miR-133b could significantly differentiate subjects with ST-elevation myocardial infarction (STEMI) from Non-STEMI. Area under the curve (AUC) for miR-133b was 0.80 with >75.6% sensitivity and specificity, AUC for miR-21 was 0.79 with >69.4% sensitivity and specificity. Our results suggest that miR-133b and miR-21 could be possible candidates of novel biomarkers in early prediction of CAD.While coronary artery disease (CAD) has become a major threat worldwide, the timely biomarker-based early diagnosis of CAD remains a major unmet clinical challenge. We aimed towards assessing the level of circulatory microRNAs as candidates of novel biomarkers in patients with CAD. A total of 147 subjects were recruited which includes 78 subjects with angiographically proven CAD, 15 pre-atherosclerotic normal coronary artery (NCA) subjects and 54 healthy individuals. Quantitative real-time PCR assays were performed. MiR-133b was downregulated by 4.6 fold ( Coronary artery disease has evidently become a major health burden, with life-threatening episodes representing the primary cause of morbidity and mortality in developed as well as in developing countries ,2. It haMicroRNAs are an evolutionarily conserved class of small noncoding RNAs (ncRNAs) approximately ~22 nucleotides in length which have emerged as a novel class of gene regulatory molecule . MicroRNSeveral studies impart information establishing miRNAs as key regulators of cardiovascular diseases ,15. InteThe hunt for circulatory biomarkers in the form of miRNAs has thus become an integral part of investigations in cardiovascular research due to their potential to enable early detection of the disease . Early dNinety-three consecutive subjects, aged 35–80 years, admitted to Cardiology ward, all India Institute of Medical Sciences (AIIMS), a tertiary care center, New Delhi, who underwent coronary angiography (CAG) for suspected case of coronary atherosclerosis, were enrolled in this case-control study. Out of these, seventy-eight patients were clinically suspected and angiographically proven cases of CAD with reasonable stenosis (>75%) and followed inclusive criteria. Fifteen subjects were suspected cases but angiographically declared as NCA patients. These subjects had stenosis levels between 50% to 69% and were hence labelled as patients with pre-atherosclerotic condition. Fifty-four healthy participants of similar age group, who were attendants of the patients and were clinically unsuspected as they had no signs or symptoms of CAD, were enrolled in this study as control subjects. Based on earlier literature available on similar types of studies ,22,25,26Coronary angiography was performed for all the enrolled case subjects by cannulating the coronary arteries according to Judkins, Trans-radial or Transfemoral techniques, equipment, and practices standard to AIIMS cardiac catheterization laboratory. The diagnoses of CAD as well as NCA were made and confirmed by coronary angiogram and according to the American College of Cardiology (ACC)/American Heart Association (AHA) 2007 guidelines. Accordingly, subjects having > 70% luminal narrowing in at least one of the major epicardial coronary arteries were classified as CAD patients. Subjects with < 70% stenosis in any of the major coronary artery were classified as NCA subjects. Patients were classified according to the evidence from angiography report and clinical evaluations for chest discomfort. Chest discomfort is defined according to signs and symptoms such as uncomfortable pressure, tightness or heaviness, chest pain that has radiated from chest to neck, back, shoulder, jaw or one or both the arms with relentless dyspnea called uncomfortable or shortness of breath due to exertion. CAD patients were further categorized based on clinical tests and symptoms as SA and ACS, which further includes UA or NSTEMI and STEMI based on standard ACC/AHA definitions and guidelines. CAD patients were also categorized based on angiography results as single vessel disease (SVD), double vessel disease (DVD) and triple vessel disease (TVD) with reasonable stenosis in the major epicardial coronary arteries. Patients with more than 70% of stenosis, systolic blood pressure (SBP) of more than 140 mmHg, diastolic blood pressure (DBP) of 90 mmHg and diagnosed with diabetes mellitus (DM) and hypertension were included in the study. Patients with less than 50% stenosis while having any sort of valvular disease, cardiomyopathy, severe hepatic or renal dysfunction were excluded from the main study. All the study subjects had been residents of Delhi and/or surrounding areas for the previous three decades and their lifestyle and food habits were recorded as standard set of questionnaires. Clinico-pathological history of the patients and medication records were also procured from the record section, AIIMS, New Delhi.The protocols adopted in the current study were conducted according to the ethical principles of the Declaration of Helsinki and its later amendments or comparable ethical standards. Certificates of application on the present proposal in the format of Indian Council of Medical Research (ICMR) were obtained and the study protocol was approved by Human Ethics committee, AIIMS, New Delhi and Human Ethics committee, ACBR, DU, Delhi. Human Ethics committee, AIIMS, New Delhi IEC/NP-207/2012 & RP-17/2012. Human Ethics committee, ACBR, DU, Delhi (F.50-2/Eth.Com/ACBR/169)2014. A written and signed informed consent form was obtained from all the participants of this study.The baseline clinical and laboratory characteristics of all the study subjects, selected for miRNA gene expression analysis, are listed in g for 10 min at 4 °C to remove erythrocytes, and then supernatant was subjected to high-speed centrifugation at 16,000× g for 15 min at 4 °C in order to remove additional nucleic acids attached to cell debris and to obtain platelet-poor plasma. Finally, the plasma was transferred to RNase/DNase-free tubes for further processing.Study samples were collected without revealing the identity of the subjects. All the researchers involved in handling and processing of the samples for total RNA isolation were blinded about the status of the study subjects to avoid any bias. Five milliliters of fasting venous blood was drawn in ice-cold EDTA containing tubes from the antecubital veins in the sitting position of all the study subjects in the morning before the cardiac catheterization procedure. To maintain the purity and integrity of the plasma, blood samples were handled with utmost care to avoid any potential disturbance and processed within 4 h of procurement of samples. Plasma was isolated from each of them using two-step centrifugation method. Blood samples were first centrifuged at 1200× Based on the previous studies ,16,24,27Total RNA enriched in RNA species smaller than 200 nucleotides, i.e., miRNAs were isolated from one milliliter of plasma samples using TRIzol LS reagent  as per the manufacturer’s protocol with slight modifications. We increased the incubation time of upper aqueous phase with isopropanol at −20 °C from 10 min to overnight (~15 h). In addition, we added 2.5 μL of glycogen solution (10 mg/mL concentration) and sodium acetate  in the upper aqueous phase to improve nucleic acid precipitation as plasma contains low amount of RNA. MicroRNA samples thus obtained were stored at −80 °C in RNase/DNase-free tubes until further processing.® Probes”, and “TaqMan Fast Advanced Master Mix” (Thermo Fisher Scientific) and detected using an ABI 7300 Real-time PCR system (Applied Biosystems® Life Technologies) according to the manufacturer’s protocols for the corresponding microRNAs. Amplification was performed in triplicates in 20 μL PCR reaction, including 5 μL of cDNA, 1 μL of “TaqMan Advanced miRNA Assay” for each miRNA quantification. The relative expression level of miR-133b and miR-21 were calculated using comparative CT method. The analytical detection limit of miRNAs was defined and set as a CT value of 40; miRNAs with a CT value > 40 were considered undetectable, and thus those samples were excluded from the subsequent analysis. In all experiments, miR-186-5p was used as an endogenous control  to normalize the samples. The normalization process was performed by subtracting the CT values of miR-186-5p from CT values of both the miRNAs in patients as well as in control samples according to the formulae ∆CT = CT value of miR-133b or miR-21—CT value of miR-186-5p.Each isolated RNA sample was polyadenylated and subjected to reverse transcription (RT) to prepare cDNA using the “TaqMan Advanced miRNA cDNA Synthesis Kit” (Thermo Fisher Scientific) as per the protocol provided by the manufacturer. cDNA was amplified using the miRNA specific “TaqManp-value < 0.05 was considered to be statistically significant. Normal Distribution of all the data sets were analyzed using Kolmogorov-Smirnov test, and they were found to be significant. Differences among the groups were compared using Student’s t-test. Qualitative variables were compared between the two groups using Chi-square test. The distribution of the miRNA expression in each group was presented as scatter/dot plot. One-way analysis of variance followed by Bonferroni correction for multiple comparisons was applied to assess the miRNA expression among controls and subcategories of CAD cases. Pearson’s correlation coefficient between study variables and miRNA expression in plasma were calculated along with the assessment for the significance of these correlations. The Receiver Operating Characteristic (ROC) curve analysis was done and the respective area under curve (AUC) was calculated in order to assess the predictive power of the circulatory miRNAs between CAD patients and healthy control group. The best threshold/cut-off value for each miRNA expression was identified and the sensitivity and specificity at that threshold value was determined to detect a CAD case, along with their 95% confidence intervals. Odds Ratios were calculated for study variables associated with CAD. A multivariable logistic regression analysis was carried out to assess the association of miRNAs expression with CAD, after adjusting for other study variables. Circulatory miRNAs expression data were used, and graphs were constructed using GraphPad prism version 8.1.1 (224) for Mac OS X  and reported as mean ± standard deviation.Expression data of both the miRNAs were analyzed using Stata version 15.1 for Windows , and presented as mean ± standard deviation. Quantitative variables were expressed as mean ± standard deviation and qualitative variables were expressed as proportions (%). A A total of 78 CAD patients , 15 NCA  and 54 healthy volunteers  of similar age were enrolled in the study. BMI, DM, hypertension, SBP, DBP, TC, HDL-C, LDL-C, and other treatment records were collected as shown in p < 0.0001) as compared to that in control group . The delta–delta-CT value for patients was found to be 2.21. The average delta-CT value for miR-21 was significantly lower (higher expression) in patients (5.24 ± 0.79) as compared to that in the control group (6.23 ± 0.96). The delta–delta-CT value for patients was found to be −0.99. CT values of miR-133b ranged from 26.4 to 32.2 in patient samples and 21.9 to 30.8 in control samples. CT values of miR-21 ranged from 18.9 to 23.1 in patients and 18.8 to 24.3 in control subjects. No template control (NTC) samples were used in any of the experiments where NTC samples showed a value in the range between 38.0 and 39.5. The average delta-CT values of miR-133b and miR-21 of patient and control groups are shown in In our study, we found the average delta-CT value for miR-133b to be significantly higher (lower expression) in patients with CAD , as shown in An altered expression pattern was observed in both the miRNAs in plasma samples of patients with subcategories of ACS group such as UA or NSTEMI (UA/NSTEMI) and STEMI, with STEMI being a more severe condition than NSTEMI. MiR-133b was able to significantly differentiate UA/NSTEMI  from STEMI (average ΔCT 16.21 ± 1.55) subjects, p value = 0.0085 and SVD (15.88 ± 1.39); p value = 0.6005, where it could not reach a significant level. This suggests a negative correlation between levels of plasma miR-133b and severity of the disease. MiR-133b expression level was also significantly altered between DVD and TVD subjects . Similarly, miR-21 expression was significantly higher in TVD (4.66 ± 0.67) than those of single vessel (5.34 ± 0.63); p value = 0.0384 or DVD (5.27 ± 0.44); p value = 0.0067. Thus, this indicates a positive correlation in the expression level of miR-21 with severity level of the disease. However, no significant difference was observed in miR-21 expression level between SVD and DVD .To evaluate the relationship of plasma miR-133b and miR-21 with CAD, we further performed a sub-analysis to determine the association of these two miRNAs to the CAD severity based on angiographically proven categories as shown in n = 22) and downregulated by 4.93 fold in CAD patients without DM (n = 56). Similarly, miR-21 was observed to be upregulated by 1.82-fold in CAD patients with DM (n = 14) and upregulated by 2.04 fold in CAD patients without DM (n = 48).We examined the expression profile of miR-133b and miR-21 both in CAD patients with DM and without DM. MiR-133b was observed to be downregulated by 3.91 fold in CAD patients with DM (To further investigate the applicability and possibility of these circulatory miRNAs (miR-133b and miR-21) as novel and potential diagnostic biomarker of CAD, we performed area under the receiver-operating-characteristic (ROC) curve analyses. A relatively good predictive value/area under curve (AUC > 0.75) was obtained in CAD patients. For instance, the ROC curve analysis a showed Likewise, the AUC value for miR-21 b was 0.7p ≤ 0.0001 and 0.0002, respectively. This was evident from the area under ROC curve (AUC) results; ROC = 0.916  for ACS vs. healthy subjects, 0.7464  for SA vs. healthy subjects, thus showing the diagnostic potential of miR-133b to accurately discriminate ACS and SA subjects from healthy subjects.ROC analysis was carried out to evaluate the discriminatory power of miR-133b for various categories or events of CAD such as SA and ACS in comparison to control subjects as well as pre-atherosclerotic condition such as NCA subjects, p = 0.0001) and thus can discriminate the ACS category from NCA. Interestingly, miR-133b could significantly differentiate pre-atherosclerotic NCA disease subjects from healthy individuals (p = 0.0446). This gives the idea that miR-133b gives a potent advantage to predict the disease condition in pre-atherosclerotic stage itself prior to severe and late-stage conditions such as ACS. MiR-133b could also significantly discriminate between ACS and SA subjects (p = 0.0063). However, in our study, a significant difference in miR-133b expression was not observed between SA and NCA (p = 0.2615).miR-133b expression was also significantly altered between ACS subjects as compared to pre-atherosclerotic NCA disease subjects (p ≤ 0.0001), as well as between SA and healthy individuals (p ≤ 0.0001), which was also evident from the area under ROC curve (AUC) results; ROC = 0.867  for ACS vs. healthy subjects and 0.7801  for SA vs. healthy subjects, thus showing its diagnostic potential to discriminate ACS and SA from healthy subjects There was a significant difference in miR-21 expression between ACS and healthy individuals (p = 0.0004) as well as SA subjects as compared to NCA , and thus suggests that miR-21 can discriminate between ACS and NCA subjects as well as between SA and NCA. Likewise, miR-21 could also significantly discriminate ACS from SA category (p = 0.0410). However, a significant difference in miR-21 expression was not observed between NCA and healthy individuals (p = 0.6522).A significant altered expression of miR-21 was observed in ACS as compared to NCA  but positively correlated with 133b . LDL and HDL showed positive correlation to miR-21 with no statistical significance  and negative correlation to miR-133b with no statistical significance .Pearson correlation analysis was performed for positive or negative association between miR-133b, miR-21 and classical risk factors. Both the miRNAs showed negative correlation to each other, p < 0.05). Multivariable logistic regression analysis revealed a significant association of both the miRNAs with CAD even after adjusting for obesity and age as confounding variables. MiR-133b (Adjusted odds ratio (OR) 11.91 (3.90–33.96) at 95% CI, p < 0.05), miR-21 (Adjusted OR 6.83 (2.31–20.22) at 95% CI, p = 0.001), Obesity (Adjusted OR 6.97 (1.86–26.19) at 95% CI, p = 0.004) and age (Adjusted OR 1.05 (0.99–1.11) at 95% CI, p = 0.93) were involved in the regression model. This suggests that both the miRNAs are independently associated with the risk of CAD.As shown in Despite tremendous advancements in treatment therapies and care, CAD is still a major cause of heart attacks and contributor in mortality rates globally ,28. MicrMicroRNAs, as endogenous molecules, have gained particular attention due to their significant role as micromanagers in gene regulation. Certain tissue- and “cell-specific” miRNAs have been clearly shown to be expressed in/and associated with cardiovascular settings and involve in maintaining vascular integrity . EmerginThe role of circulatory miR-133b and miR-21 in plasma samples of CAD subjects is unknown and scarcely studied. In the present study, we have demonstrated the association of altered expression profile of circulatory miR-133b and miR-21 in patients with CAD in comparison with healthy individuals. We also tried to demonstrate the potential of both the miRNAs to discriminate atherosclerotic condition (CAD) from pre-atherosclerotic condition (NCA). MicroRNA-133b (miR-133b) has been predominantly appraised as a “muscle-specific” miRNA , and thuIn light of the above studies, wherein, using animal models, tissue samples and “cardiac-specific” cell lines, the utility of various circulatory miRNAs was shown to have significant diagnostic potential for cardiovascular complications, we decided to ascertain the potential role of miR-133b and miR-21 as non-invasive diagnostic biomarkers in CAD. Previously, Ai J et al. (2010) showed miR-133b expression in 93 AMI patients’ samples, but the results were not statistically significant . SimilarUsing 78 CAD patients, our study provides evidence for the significant association of miR-133b (negative correlation), and miR-21 (positive correlation) to the risk of CAD. We found that miR-133b expression in plasma samples of CAD patients was markedly downregulated by 4.63 fold, whereas miR-21 expression was upregulated by ~2 fold as compared to healthy individuals. The results for miR-133b expression pattern were in line with the observations of recently reported studies ,45,46,47p = 0.0446) differentiate NCA condition from healthy subjects, with a fold difference of 2.08. This suggests that even when the NCA condition is considered or close to the healthy stage, there were significant differences in the expression of miR-133b in NCA subjects as compared to healthy subjects, possibly due to the extent of stenosis (50%–69%). These results may suggest that miR-133b can predict pre-atherosclerotic condition from rest of the stages of CAD. MicroRNA-21 (miR-21) also showed a clear difference between healthy subjects and the pre-atherosclerotic NCA condition, with a fold difference of 1.12. In conclusion, pre-atherosclerotic subjects showed similar trends of expression of both the miRNAs as shown in patients with higher stenosis. These results should be further confirmed using a large data set, but this surely provides an indication that miR-133b and miR-21 have potential to differentiate an early stage or pre-atherosclerotic stage (NCA) from CAD comprising the SA and ACS condition. In the present study, we also found that the expression profile of both the miRNAs starts changing before the actual severity level of the disease comes into the process. Based on our results, the expression pattern of both the miR-133b and miR-21 starts changing even in NCA subjects, who are referred to as pre-atherosclerotic subjects, wherein the extent of stenosis can range from 50% to 69%. NCA was considered to be a pre-atherosclerotic stage where the subjects are otherwise considered to be angiographically proven normal subjects with no significant stenosis in any of the major coronary arteries. MicroRNA-133b (miR-133b) could significantly  condition (negative correlation) compared to higher expression in DVD and SVD. Likewise, the highest expression level of miR-21 was observed in TVD (positive correlation) as compared to that in subject with SVD and DVD conditions. To further elucidate the association of miR-133b and miR-21 with ACS, we attempted to investigate the potential role of both the signatures in discriminating different pathological conditions and categories within ACS, i.e., STEMI and UA/NSTEMI. We observed a significantly lower expression pattern of miR-133b in most severe STEMI condition as compared to the other category (UA/NSTEMI), thus indicating its potential to differentiate between the two categories. In our study, both the categories showed an altered expression of miR-21, but a significant difference was not observed. Until now, very few researchers have studied the role of miR-133b and miR-21 in the ACS condition specifically in its subcategories (STEMI and UA/NSTEMI). Diagnostic potential to identify the discriminatory/predictive power of miR-133b and miR-21 was assessed by means of area under ROC curve analysis. MiR-133b and miR-21 were found to be highly predictive as potential biomarkers of CAD. Diagnostic potential and predictive power of miR-133b and miR-21 with respect to CAD was as high as 80% and 79% respectively, with an upstanding sensitivity and specificity. Moreover, ROC results showed that both the miRNAs could predict ACS condition more prominently and precisely compared to SA and other conditions, thus suggesting their high utility as non-invasive diagnostic biomarkers in the CAD condition.The current study provides the first clinical evidence of circulating miR-133b and miR-21 as novel non-invasive predictive biomarker in diagnosing CAD in Indian subjects. Our study consists of study subjects from the north India region and surrounding areas. As this is a single/limited region study, a multiregional or multicentric approach may provide a broad picture which can further enhance the validity of the prospective results. So far, not many efforts have been directed towards establishing a significant association between some of the circulatory candidate miRNAs and the severity of CAD when developing a non-invasive diagnostic method that can help in reducing coronary complications. Considering CAD as a multifactorial and multigenic disease driven by various genetic and non-genetic factors, observations from the current study appear to support the assumption that miRNA signatures have important implications on CAD and its different stages.Based on the results from the current study, miR-133b and miR-21 are significantly associated with the risk of CAD and different subcategories. Both the miRNAs have a potent ability to discriminate and predict various stages of the disease from pre-atherosclerotic to its severe atherosclerotic form. Observations from the current study, although exploratory, shed some light on the pathophysiological role of miRNAs, thus propounding their potential inclusion in coronary artery complications. A large cohort of patient studies are still warranted in order to unequivocally establish the potential diagnostic utility of miR-133b and miR-21 in rapid diagnosis of CAD patients and their subcategories, where conventional biomarkers, such as cardiac troponins are less reliable or are of invasive nature. In conclusion, together with the previous reports and the findings from the current study, the advantages provided by miRNA signature shows their potential to be utilized as biomarkers of early prediction of coronary complications in patients with CAD, thus indicating their integration with other conventional parameters such as cardiac troponin in order to improve the diagnosis of vascular diseases such as CAD.


Cryptococcus neoformans complex, previously characterized by Amplified Fragment Length Polymorphism (AFLP) and sequencing of the ITS1-5.8S-ITS2 region: (i) an expanded database was built with 26 isolates from the main Cryptococcus species found in our setting  and (ii) peak analysis and data modeling were applied to the protein spectra of the analyzed Cryptococcus isolates. The implementation of the in-house database did not allow for the discrimination of the interspecies hybrids. However, the performance of peak analysis with the application of supervised classifiers  in a two-step analysis allowed for the 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. In addition, PCA analysis prior to support vector machine (SVM) provided 98.45% correct discrimination of the three analyzed species in a one-step analysis. This novel method is cost-efficient, rapid and user-friendly. The procedure can also be automatized for an optimized implementation in the laboratory routine.Matrix-assisted laser desorption–ionization/time of flight mass spectrometry (MALDI-TOF MS) has been widely implemented for the rapid identification of microorganisms. Although most bacteria, yeasts and filamentous fungi can be accurately identified with this method, some closely related species still represent a challenge for MALDI-TOF MS. In this study, two MALDI-TOF-based approaches were applied for discrimination at the species-level of isolates belonging to the  Cryptococcus has classically comprised two sibling species with great clinical importance: Cryptococcus neoformans and C. gattii, the causative agents of cryptococcosis. Whilst the C. neoformans complex has been associated with meningitis in immunosuppressed patients, C. gatti has been shown to cause disease in both immune competent and immunocompromised populations [Cryptococcus species and molecular types [C. neoformans; AFLP2/VNIV for C. deneoformans, AFLP3/VNIII for the interspecies hybrid C. neoformans x C. deneoformans; and AFLP4/VGI, AFLP5/VGIII, AFLP6/VGII, AFLP7/VGIV and AFLP10/VGIV-VGIIIc for the C. gattii complex [Cryptococcus spp. identification, its high requirements in hands-on time and expertise has led to the evaluation of alternative tools.The genus ulations ,2,3. Speulations ,5,6,7. Tulations . The avaulations , amplifiulations , multi-lulations , and mululations  has alloar types ,14. Geno complex ,16. ThesCandida genus have been shown to be easily identified at the species level either from single colonies or directly from clinical samples using MALDI-TOF MS [Candida yeasts still represent a challenge for this technology, especially when applied to the identification of genera poorly represented or lacking in the commercial databases [C. neoformans and C. gatti complexes [Matrix-assisted laser desorption–ionization/time of flight mass spectrometry (MALDI-TOF MS) has emerged as a promising technology for the rapid and reliable identification of yeasts ,18,19. II-TOF MS . Howeveratabases . In thisatabases . Althougomplexes ,23, the C. deneoformans, C. neoformans and the interspecies hybrids was developed: protein spectra from Cryptococcus isolates were analyzed and classified using different algorithms in order to find species-specific peaks that allowed their differentiation. In addition, a database was built using well characterized isolates for the identification of the Cryptococcus spp. isolates using the Biotyper system developed by Bruker Daltonics  and the identifications obtained were compared with the peak-analysis method.In this study, a novel MALDI-TOF-based methodology for the discrimination between Cryptococcus spp. isolates from clinical samples (n = 70) belonging to 67 patients admitted to Hospital Gregorio Marañón  from 1994 to 2007 (n = 44) were used to evaluate the commercial database alone or in combination with the in-house library. For the peak analysis approach, protein spectra from 65 Cryptococcus isolates were included since the remaining isolates could not be recovered from the frozen backup after MALDI-TOF MS identification.We retrospectively selected 70  to 2007 . IsolateTo ensure the purity, the isolates were grown on Columbia agar supplemented with 5% of sheep blood plates and incubated at 35 °C for 24 h. All isolates were previously identified by the DNA sequencing analysis of the ITS1-5.8S-ITS2 region . MoleculCryptococcus spp. isolates were analyzed using an LT Microflex benchtop MALDI-TOF mass spectrometer  using default settings. FlexControl 3.3 and MALDI Biotyper 3.1 were applied for spectra acquisition and comparison with the reference spectra present in the BDAL database, updated with 8223 MSPs . This database contains 12 reference MSPs from C. neoformans and 7 from C. deneoformans. Twenty-six isolates from the hospital collection were selected as representatives of the Cryptococcus species from this study and included in the in-house library . Then, the expanded in-house HGM library was challenged in combination with the commercial database with the remaining 44 isolates.Seventy The sample processing method applied consisted of a mechanical disruption step followed by a standard protein extraction . BrieflyThe identifications provided by MALDI-TOF MS were compared at the species level with those provided by AFLP analysis regardless of their score value (Table 1). Moreover, score values ≥2.0 were considered as “high-confidence” scores and those ≥1.7 as “low-confidence” ones. Score values below 1.6 were only considered as “very low-confidence” identifications—but reliable—when consistent along the four top identifications, otherwise they were considered as “not reliable”.Cryptococcus species, their protein spectra were processed using Clover MS Data Analysis software  with the parameters shown in m/z. Furthermore, spectra alignment was performed. First, the replicates from the same isolate were aligned in order to get an average spectrum. Finally, all average spectra were aligned together.For the classification of the three The rate of presence for the biomarker peaks was calculated for each species and then compared among species. The receiver operating characteristic (ROC) curve with area under the curve (AUC) higher than 0.99 were used as quality indicator to measure the sensibility and specificity of a selected biomarker.Cryptococcus isolates, processed as described in Once the putative biomarkers were selected and analyzed, a peak matrix was built containing all the aligned spectra from all Cryptococcus species. The first one was a two-step method in which the discrimination of C. deneoformans from the other two species was performed as a first step and it was replicated by means of two supervised machine learning algorithms on the same peak matrix: partial least squares discriminant analysis (PLS-DA) and support vector machine (SVM). Results were validated using k-fold cross validation method.Over the peak matrix, two approaches were applied in order to discriminate the three C. neoformans from the interspecies hybrids. Again, PLS-DA and SVM were applied to this second peak matrix to replicate the classification and k-fold cross validation was applied. The two-step method was further improved by the removal of peaks that did not provide enough discrimination.In the second step, a new peak matrix was performed in order to achieve a better discrimination of Cryptococcus species at the same time was tested. In this case, only one peak matrix with spectra from the three species was built and five species-specific biomarkers were included. The alignment and processing parameters were the same as in the two-steps approach. As in the previous cases, PLS-DA and SVM analysis was performed and results were validated using k-fold confusion matrix.Finally, in order to simplify the workflow, a one-step method was assayed so that the capacity of the algorithms to discriminate the three Cryptococcus isolates—C. neoformans (n = 12), interspecies hybrids (n = 10) and C. deneoformans (n = 4)—were processed according to the manufacturer’s instructions and added to the in-house database  as individual main spectra (MSPs). Following the manufacturer’s recommendations, at least ten MSPs from each species were planned to be included in the in-house library. However, this figure was modified according to the availability of isolates in our local collection.Twenty-six Cryptococcus isolates were processed as explained below and then spotted onto eight positions in the MALDI target plate and each position was read three times. Twenty-four protein spectra were thus achieved, 20 of which had to be identical in order to be accepted by the software  as an MSP and then added to the extended library.The procedure for adding new entries to an in-house library has already been described . BrieflyThe hospital Ethics Committee approved this study and gave consent for its performance (Code: MICRO.HGUGM.2017-003). Since only microbiological samples were analyzed, not human products, all the conditions to waive the informed consent were met.C. neoformans, n = 34; 49%), followed by AFLP3  and AFLP2 .The genotyping of the isolates detected three different genotypes. The most common genotype was AFLP1/1B (C. neoformans isolates (81.8%) and 1/3 C. deneoformans isolates (33.3%); the remaining C. neoformans isolates—n = 4—could not be reliably identified and for 2 C. deneoformans isolates, MALDI-TOF MS did not provide species identification (n = 19) was not achieved using the commercial database due to the lack of representation of this microorganism. These isolates were identified as C. neoformans complex in nine cases , as C. deneoformans in seven cases  and as C. neoformans in three cases (score > 1.7)—The application of MALDI-TOF MS and the commercial database allowed the correct identification of 18/22 fication . The ideC. neoformans isolates (4.5%) were correctly identified at the species level with high-confidence score values (≥2.0) whilst 29.5% of the samples—13—were correctly identified with low-confidence scores (≥1.7)—Only two C. neoformans and C. deneoformans isolates were correctly identified by MALDI-TOF MS at the species level  score of 96.92% for PLS-DA, which performed over the peak matrix with 10 biomarkers and 98.46% for the analysis with five biomarkers. However, the SVM algorithm achieved 100% discrimination in both cases when PCA was applied .C. neoformans and the interspecies hybrids . Furthermore, this second dendrogram was validated by PLS-DA and SVM algorithms. K-fold (k = 10) was applied achieving 95.55% accuracy in both analyses.A second dendrogram was performed using hierarchical clustering analysis. It showed two well defined clusters for  hybrids . In thisCryptococcus species simultaneously. PLS-DA analysis could not classify correctly the three species at the same time due to the low k-fold (k = 10) values obtained. However, the PCA performance prior to SVM allowed 98.46% correct classification of the three Cryptococcus species  were detected in 100% of the spectra from this species .Therefore, a novel approach consisting of the analysis of the protein peaks present in the MALDI-TOF MS spectrum has been developed in this study. A list of 10 biomarker peaks was achieved as the input for the species classification. The implementation of PLS-DA analysis in a two-step approach allowed the discrimination of C. deneoformans isolates and of a high rate of C. neoformans and interspecies hybrids despite their genetic variability, demonstrating the robustness of this study. In addition, this analysis provided a set of species-specific peaks for the Cryptococcus species within the C. neoformans complex that may be detected by visual inspection, representing a rapid and inexpensive approach for their discrimination.In order to simplify the analysis, a one-step method was applied to classify the three species simultaneously. In this case, PLS-DA provided correct classification in less than 75.0% of the cases but the application of SVM after PCA analysis allowed 96.92% correct discrimination of the analyzed isolates. This algorithm allowed for the correct discrimination of all Cryptococcus spp. isolates. Although the updated commercial library provided correct species-level identification for a high number of C. deneoformans and C. neoformans isolates, the identification of these species was missing or not reliable in 20.5% and 18.2% of the cases, respectively. Moreover, the detection of the interspecies hybrids was not possible with the Biotyper database. However, the expanded in-house library allowed for the correct species-level identification for all C. deneoformans and C. neoformans, either by conventional identification with MALDI-TOF MS or by peak analysis. The interspecies hybrids required hierarchical clustering for their correct identification since their close relatedness with the other species made it difficult for MALDI-TOF MS to differentiate them from the other two species in a routine manner. This novel approach and the visual detection of species-specific peaks can be implemented in microbiology laboratories where ad hoc libraries are not available.Our results demonstrate the usefulness of MALDI-TOF MS and peak analysis when applied in the microbiology laboratory for the rapid and reliable identification of


The novel coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading to a worldwide pandemic. Except representative manifestation of pneumonia and acute respiratory symptoms, COVID-19 patients have also shown different levels of liver injury or liver dysfunction. The aim of our study was to explore the probable clinical severity and mortality of COVID-19 patients and their liver dysfunction.A combination of computer and manual retrieval was used to search in Medline through PubMed, EMBASE and Web of Science. Review Manager 5.3 software was used to examine the heterogeneity among the studies and to calculate the combined effect value . Subgroup analysis, sensitivity analysis, and publication bias test were also performed.P = 0.0002). There was a significant association between AST and severity of COVID-19 with a pooled OR of 4.48 , and a pooled WMD of 3.35 . In addition, there was a significant difference between TBIL and severity of COVID-19, with a pooled OR of 1.91 , and with a pooled WMD of 1.18 .We found a significant connection between liver dysfunction and mortality of COVID-19 patients with a pooled OR of 1.98 (95% CI 1.39–2.82; The mortality and severity of COVID-19 patients are significantly associated with liver dysfunction. The non-survivors and severe COVID-19 patients have elevated serum AST levels than the survivors and non-severe COVID-19 patients. The results of this study form a basis for better clinical liver management of patients with COVID-19. Among the 99, 43 (43.4%) had elevated aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) [n = 279) and severe (n = 269) patients (P = 0.683) [. retrospectively studied 135 hospitalized COVID-19 patients and reported that there were no significant differences in ALT (P = 0.73) and total bilirubin  between mild cases (n = 95) and severe cases (n = 40) [Coronavirus (CoV) is an enveloped positive single-stranded RNA virus. It is widely distributed in humans and animals. It is known to cause human respiratory infections . The ongCOVID-19 . A recenCOVID-19 . Chen et= 0.683) . Similar(n = 40) . These cStudies involving SARS-CoV-2 or COVID-19 or liver injury/dysfunction were included. In order to find relevant original articles, we conducted a comprehensive search in the database, involving Medline through PubMed, EMBASE and Web of Science, and using the following words: "COVID-19", "SARS-CoV-2", "Wuhan virus", "Chinese virus", "novel coronavirus 2019", "2019 nCoV", "Wuhan coronavirus", ``2019 Coronavirus", "Characteristics", "Characteristics" and "Liver". As of April 17, 2020, the papers have been searched in the language range. We also refer to the recognized literature to find other qualified research subjects. We first screened the article title and abstract, as well as publications that may involve data on COVID-19 or SARS-CoV-2 and liver injury/dysfunction.This study had no national restrictions. All studies reporting on COVID-19 non-survivors/survivors, non-severe/severe and laboratory confirmed COVID-19 patient data were included in the study. Moreover, the studies had to be limited to humans, include raw data, be published in English and be in either abstract form or full text. Repeated studies, letters, case reports, abstracts, and comments were excluded from the study. Data collected from each study included the year of publication, name of first author, sample size, and age of COVID-19 patients.I2 index. I2values less than 25%, 25–50%, 50–75% and 75–100% were homogeneous, or had low, medium, and high heterogeneity levels, respectively. The random effect model (REM) was applied if the I2 value is > 50% while the fixed effect model (FEM) was applied if the I2 value is < 50%. The combined odds ratio (OR) and weighted mean difference (WMD) of different studies with corresponding 95% confidence intervals (CI) were used to assess the relationship between liver dysfunction and COVID-19. The sensitivity analysis was repeated to evaluate the impact of each study in the analysis by subsequently excluding different individual studies each time. Based on the method of Hozo et al. [The quality of each study was evaluated using the Newcastle–Ottawa scale while the meta-analysis was conducted through the Review Manager 5.3 software. Heterogeneity was evaluated by calculating the o et al. , the meaFive hundred and eighty-five relevant articles and one from the reference list were identified. Exclusion of duplicates brought down the number to 326 studies. The 326 studies were further screened if they meet the inclusion criteria. This resulted in the exclusion of 270 studies. Forty-three more studies were excluded after scanning of the entire body text of the 56 remaining studies. The remaining 13 articles meet the eligibility criteria , 13–21. P = 0.0002). The pooled data were calculated under the FEM as a low heterogeneity within the studies. Pooled results of five studies revealed that AST was significantly lower in COVID-19 survivors   . However, ALT was significantly lower in patients with non-severe COVID-19  , and a pooled WMD of 3.35  , and a pooled WMD of 1.18  . However, when either Chen’s or Wang’s study was eliminated, the results significantly affect the pooled outcomes (I2 dropped from 36 to 25%). Similarly, when Chen’s study was removed from the AST group, the I2 dropped from 93 to 70%. In the same line, the severity of COVID-19 in AST (events) had moderate heterogeneity (I2 = 61%). However, when Li’s study was eliminated, the I2 dropped from 61 to 49%. Similarly, when Zhang’s study was removed in the AST (levels) group, the I2 dropped from 97 to 79%. When Zhang’s study was removed in the AST (events) group, the I2 dropped from 71 to 28%. In the TBIL (levels) group, removal of Zheng’s study led to reduction of the I2 from 71 to 67%. Cognizant of this, it was hypothesized that the studies of Zhang et al. 2020 and Zheng et al. 2020 were the sources of heterogeneity in this meta-analysis. The Egger’s regression test (P > 0.05) further revealed that the meta-analysis had no significant publication bias.Subgroup analysis was used to reveal the sources of heterogeneity. Age and the number of non-severe patients were found to be potential sources of heterogeneity Table . HoweverHerein, the abnormal liver function was associated with mortality and severity of COVID-19. Subgroup analysis conducted to identify sources of heterogeneity further revealed that the overall outcome was acceptable. Similarly, sensitivity analysis revealed that when any study was excluded or REM was converted to FEM, the overall outcome was acceptable. These results strongly suggested that there in a correlation between liver injury and COVID-19.. [Although it has been observed that the most severe and fatal cases of COVID-19 occur in the elderly individuals with liver injury, the relevant pathophysiology is still unclear. Xu et al  reported.  reported. 2020 reported that the survival rate of patients aged above 75 years was significantly lower than that of young patients [Patients aged above 65 years have higher comorbidities, more severe symptoms, and are more prone to multiple organ involvement and death compared to the younger patients . For exapatients . Factorspatients . CognizaPrevious studies quantify the effects of COVID-19 on the digestive system and chronic liver disease , 28. WhiThe mortality and severity of COVID-19 patients are significantly associated with liver dysfunction. The non-survivors and severe COVID-19 patients have elevated serum AST levels than the survivors and non-severe COVID-19 patients. The results of this study form a basis for better clinical liver management of patients with COVID-19.


These data outperformed the commercial FAA-3-50 membrane sold by Fumatech in Germany. Based on the optimized properties, membrane electrode assembly using XAc-PPO70_25 revealed excellent cell performance (maximum power density: 239 mW/cm2 at 0.49 V) than those of commercial FAA-3-50 Fumatech anion exchange membrane (maximum power density: 212 mW/cm2 at 0.54 V) under the operating condition of 60 °C and 100% RH as well. It was expected that PF-XAc-PPO70_25 could be an excellent candidate based on the results superior to those of commercial membranes in these essential characteristics of fuel cells.In this study, novel crosslinked pore-filling membranes were fabricated by using a centrifugal force from the cylindrical centrifugal machine. For preparing these crosslinked pore-filling membranes, the poly(phenylene oxide) containing long side chains to improve the water management (hydrophilic), porous polyethylene support (hydrophobic) and crosslinker based on the diamine were used. The resulting membranes showed a uniform thickness, flexible and transparent because it is well filled. Among them, PF-XAc-PPO70_25 showed good mechanical properties (56.1 MPa of tensile strength and 781.0 MPa of Young’s modulus) and dimensional stability due to the support. In addition, it has a high hydroxide conductivity (87.1 mS/cm at 80 °C) and low area specific resistance (0.040 Ω·cm As climate change accelerates, the development of green energies has attracted significant attention ,2. AmongTherefore, improvement of conductivity and stability through various techniques is important in the AEMFC ,12,13,14The present work focused on the improvement of water management, conductivity, mechanical and chemical stability of anion exchange membranes based on the pore-filled membrane using the poly(phenylene oxide) with long side chain and quaternary ammonium group through Friedel crafts acylation reaction, and polyethylene (PE) support .N2) may remain [Even with the above solution, chemical stability problems due to (i) Hofmann elimination (E2), and (ii) nucleophilic substitution  were obtained from Sigma Aldrich Korea . Methyl alcohol (MeOH), N-methyl-2-pyrrolidone (NMP) and potassium hydroxide (KOH) were purchased from Daejung Chemical . The polyethylene (PE) support was provided from W-SCOPE KOREA .Poly (PPO) was purchased from Asahi Kasei Corp . 6-bromohexanoyl chloride, aluminum chloride, 1,2-dichloroethane anhydrous, trimethyl ammonium solution (45 wt% in DI water) and 3, 4.27 g, 32.04 mmol) was dispersed in the 1,2-dichloroethane anhydrous solvent with nitrogen gas at the one neck round bottom flask, and then, 6-bromohexanoyl chloride  was dropped slowly in the dispersed solution using a dropping funnel. The reagent solution was obtained after 30 min. Then, this solution was inserted to the PPO solution using dropping funnel at the ice bath. After fully dropping the reagent solution, the final solution was maintained at the room temperature for 6 h, and the solution was precipitated using methyl alcohol, the Ac-PPO obtained was washed several times in the same non-solvent. Finally, the product was dried in a vacuum oven at 60 °C for 24 h, resulting in Ac-PPO70 at a yield of 94.71%.The acylated poly(phenylene oxide) (Ac-PPO) was synthesized in a 3-neck round bottom flask with dropping funnel. The PPO solution was prepared by dissolving poly  in 1,2-dichloroethane anhydrous solvent. At the same time, the reagent solution for Friedel-Crafts acylation reaction was prepared using 6-bromohexanoyl chloride and fecalaluminum chloride. More details, fecalaluminum chloride  to modify into conducting group for 24 h. This is because the modifying reaction is a heterogeneous condition, it needs enough replacing time. The pore-filled membrane containing conducting group was subjected to alkalization in 1M KOH solution for 24 h at room temperature. The OH– exchanged membranes were washed several times with deionized water to remove any residual KOH and stored in DI water until the characterization process. The pore-filled membrane using PE substrate is named as PF-XAc-PPOX_Y membrane, where, X represents the degree of substitution (DS) of quaternary ammonium group and Y represent the degree of crosslinking, respectively. For example, PF-XAc-PPO70_25 is prepared as a pore-filling membrane using a PE support in a 25% crosslinked solution of an Ac-PPO polymer having a degree of substitution of 70%.1H-NMR spectra of Ac-PPO were obtained on DRX300 (300 MHz) . Chloroform-d was used as the solvent.The The morphology of the crosslinked pore-filling membranes was observed by means of a FE-SEM . The observations of the pore-filled membranes consisted of surface and cross-section assessments.HClM (M) and HClV (mL) are correspondingly the concentration and volume of the initial HCl solution. NaOHM (M) and NaOHV (mL) are the concentration and volume of standard NaOH solution used for titration, respectively. dryW (g) is the weight of the dry crosslinked pore-filling membrane.The back-titration method was used for the anion exchange membrane. The pore-filled membrane was immerged in a 0.01M HCl solution for 24 h, and the IEC value was measured by titration using a 0.01M NaOH solution. After finishing the titration process, the pore-filled membrane was removed and dried in a vacuum oven at 60 °C. The weight of the dried pore-filled membrane was then measured. Finally, the ion exchange capacity was calculated using the following equation:dryW). These were then immersed in DI water for 24 h. Excess water on the sample surface was wiped away with tissue paper and weighed (wetW). The water uptake was calculated using the following equation:The water uptake was calculated according to the weight change of the crosslinked pore-filling membrane. To determine the water uptake value, the crosslinked pore-filling membrane was cut into an appropriate size, and then weighed (λ) was determined using the following equation:The hydration number of water molecules per ionic group  of pore-filled membranes were measured by immersing the rectangle shape membranes into deionized water, and the changes of size were calculated using the following equation:The mechanical properties of the crosslinked pore-filling membrane were measured by using a universal testing machine  according to the ASTM D638 5. To confirm the mechanical properties, the tension speed was 10 mm/min.The hydroxide conductivity of the pore-filled membrane is an important factor. The hydroxide conductivity was determined using the measured value of the membrane resistance. The hydroxide conductivity tests of the pore-filled membranes were carried out at from 25 to 80 °C and at a relative humidity of 100% by the impedance method using electrochemical spectroscopy . Finally, the hydroxide conductivity was calculated using the following equation:R is the electrical resistance, and L and A are respectively the thickness and area of the membrane. The ionic area specific resistance (ASR) was calculated using the following equation:σ is the ion conductivity, T is the thickness of the crosslinked pore-filling membranes and σ is the hydroxide conductivity at 80 °C.The alkaline stability of the crosslinked pore-filling membranes was investigated by soaking the OH− form membranes into 1M KOH solution in 60 °C for 800 h to evaluate the changes in ionic conductivity. Before measurements, each membrane was washed with deionized water several times to remove the free KOH inside the membrane. The ionic conductivity of each membrane was measured in high purity deionized water at room temp.2 and O2 feed flow rates of 300 mL/min and 300 mL/min respectively. The catalyst slurry was prepared using a mixture of Pt/C catalyst , 2-propanol and ionomer solution  by ultra-sonicating method. The CCMs  were prepared by spraying the slurry on the membrane (1.0 mg Pt/cm2) with an air spray gun. The MEA (Membrane Electrode Assembly) was sandwiched between gas diffusion layer  and gasket  and the effective area was 9 cm2.Single cell performances using the membranes were analyzed at 60 °C under 100% RH and H1H-NMR spectroscopy.1H-NMR spectrum of the Ac-PPO polymer. In the 2–) and the bromine . In addition, the degree of substitution was determined from the integral area values ratio of the peak at 6.45 and 6.06 ppm [6.06 ppm .To obtain the chemical stability of the pore-filled membrane, crosslinking was performed using a halogen element and a diamine crosslinking agent, and the degree of crosslinking was adjusted through the amount of the crosslinking agent. In In the anion exchange membrane fuel cell, since local hydration of the too thin electrolyte membrane less occurs, the resistance increases, resulting in performance degradation . For thaAs can be seen from To improve the durability of the membrane electrode assembly (MEA), the mechanical strength of the crosslinked pore-filling membrane is an important factor. In the case of XAc-PPO70_100, its physical properties were so weak that it could be crushed when touched. However, the crosslinked pore-filling membranes showed excellent mechanical properties. In particular, PF-XAc-PPO70_25 showed a tensile strength of 56.1 MPa, an elongation at break of 32.7%, and a Young’s modulus of 781.0 MPa. It was confirmed that the elongation gradually decreased as the degree of crosslinking increased as reported by other studies ,39,40,411H-NMR spectra and was similar to the experimental value. The IEC values of all the PF-XAc-PPO70_Y showed lower than the XAc-PPO70_100 relatively. The lower IEC values observed for the crosslinked pore-filling membranes were ascribed to the porous PE support. In the case of IEC, since the unit is “meq/g”, it is affected by weight. However, there is no ion conducting group per unit weight in the case of the porous support, so the IEC of crosslinked pore-filling membranes with the support are relatively low [In the case of XAc-PPO70_100, IEC was calculated as 2.96 meq/g based on the vely low . As can The water uptake and swelling ratio of the crosslinked pore-filling membranes in hydroxide form at 25 and 80 °C are depicted in In addition, water management of the electrolyte membrane is essential for fuel cell performance and durability of the entire system in AEMFC . At thisThe hydroxide conductivity is an important factor on which the fuel cell performance and efficiency depend. The hydroxide conductivities of the crosslinked pore-filling membranes with different degree of crosslinking were measured at 25, 40, 60 and 80 °C. As shown in Moreover, the conductivity under humidified conditions did not differ significantly when compared to the conductivity in water . Continua values of crosslinked pore-filling membranes are in the range of 9.85 to 7.99 KJ/mol. These values are comparatively better than those values of previously reported anion exchange membranes .2 or less. The ionic ASR values for each crosslinked pore-filling membrane can be found in 2 [Recently, the area specific resistance (ASR) is considered as important data for fuel cells as well as hydroxide conductivity. As a clear example of this, the US Department of Energy is targeting an ASR of 0.04 Ω·cmund in 2 .N2 reaction and the Hofmann elimination reaction. An alkaline stability was tested with the crosslinked pore-filling membranes in 1M KOH solution at 60 °C for 800 h.The chemical stability as known as alkaline stability of anion exchange membrane under alkaline medium conditions is related to the performance stability of AEMFC. In an alkaline atmosphere, the polymer main chain or conducting group may be degraded due to the SAs can be seen in the This is believed to be due to the fact that water management is possible due to a relatively small number of crosslinking, and thus the attack on the main chain is less .2/O2 feed gas under 100% RH and 60 °C conditions. As can be seen in 2 at ad current density of 549 mA/cm2. These results were much higher than those obtained from the commercial Fumatech FAA-3 membrane (392 mA/cm2 of current density and 212 mW/cm2 of maximum power density).  through the Friedel-craft acylation reaction. At the same time, crosslinking was performed using a diamine crosslinker, and the porous PE support was well filled using a centrifugal force. All of the crosslinked pore-filling membranes exhibited good water uptake, swelling ratio, mechanical strength, hydroxide conductivity and chemical stability. Specifically, PF-XAc-PPO70_25 showed a hydroxide conductivity of 87 mS/cm at 80 °C in the DI water and 79.3 mS/cm at 80 °C in the humidity condition. The single cell performance of a membrane electrode assembly (MEA) employing the PF-XAc-PPO70_25 was found to be comparable to that employing a commercial membrane (FAA-3-50) due to good water management and physicochemical stability. Therefore, based on the above data, we believe that our crosslinked pore-filling membranes are promising candidates for anion exchange membrane fuel cell (AEMFC).


Hypoxia plays a significant role in solid tumors by the increased expression of hypoxia-inducible factor-1α (HIF-1α), which is known to promote cancer invasion and metastasis. Cancer-cell invasion dynamically begins with the degradation of the extracellular matrix (ECM) via invadopodia formation. The chemical substrates that are utilized by hypoxic cells as fuel to drive invadopodia formation are still not fully understood. Therefore, the aim of the study was to maintain MDA-MB-231 cells under hypoxia conditions to allow cells to form a large number of invadopodia as a model, followed by identifying their nutrient utilization. The results of the study revealed an increase in the number of cells forming invadopodia under hypoxia conditions. Moreover, Western blot analysis confirmed that essential proteins for hypoxia and invadopodia, including HIF-1α, vascular endothelial growth factor (VEGF), metallopeptidase-2 (MMP-2), and Rho guanine nucleotide exchange factor 7 (β-PIX), significantly increased under hypoxia. Interestingly, phenotype microarray showed that only 11 chemical substrates from 367 types of substrates were significantly metabolized in hypoxia compared to in normoxia. This is thought to be fuel for hypoxia to drive the invasion process. In conclusion, we found 11 chemical substrates that could have potential energy sources for hypoxia-induced invadopodia formation of these cells. This may in part be a target in the hypoxic tumor and invadopodia formation. Additionally, these findings can be used as potential carrier targets in cancer-drug discovery, such as the usage of dextrin. Hypoxia is observed in the majority of solid tumors. In solid tumors, aggressiveness, angiogenic activity, and tumor progression (enhanced due to low-oxygen) exist in most if not all malignant cancers that are known to resist chemotherapy and radiotherapy. Studies reported that hypoxia is a driving force for breast-cancer progression ,2. PreviHypoxia in solid tumors can alter cancer-cell metabolism and contributes to therapy resistance by inducing cell quiescence . AdaptatHIF-1α can also promote metastasis, which is a critical step in all malignant cancers in which cancer cells spread away from the primary tumor to distant organs. The majority of cancer mortality is due to metastasis . It occuInvadopodia are specialized actin-rich membrane protrusions that contain actin filaments, and they are formed at the ventral surface of malignant cells, such as MDA-MB-231 breast-carcinoma cells . The MDAThe ability of malignant cells to form invadopodia usually depends on their invasiveness potential ,18,19. I2) and 0.5 mM of dimethyloxalylglycine (DMOG), a chemical compound that works as an HIF hydroxylase inhibitor that is widely used to mimic hypoxia conditions in order to use phenotype microarrays for mammalian cells.In the current study, one area that is yet to be explored is carbon-source utilization that MDA-MB-231 breast-cancer cells can consume as nutrient providers for hypoxia conditions to drive the formation of invadopodia. A recent report established that improved cancer therapy may be achieved by exploring the nutrient utilization of tumor cells . Thus, t2, 5% CO2, and balanced with N2) was examined after seeding hypoxic or nonhypoxic cells on gelatin-coated coverslips. One of the key components of invadopodia, actin, was stained, and it localized at the center of the cells , and the hypoxia chamber  for  h compared to in normoxia. However, at 12 h incubation, the expression level of HIF-1α was relatively low, yet it was still higher than that in normoxic cells  are essential for invadopodia formation in MDA-MB-231 cells . Here, wWe hypothesized that hypoxia utilizes more chemical substrates as fuel for cellular activity, such as the invasion process. To investigate the preferences of chemical substrates under hypoxia conditions in MDA-MB-231 cells, a phenotype microarray for mammalian cells was employed to understand why cancer-cell invasion was promoted under hypoxia conditions, particularly by the increase in the level of invadopodium formation, by monitoring the levels of carbon- and nitrogen-source utilization. Phenotype microarray comes in four 96-well plates precoated with 367 types of chemical substrates. A high number of cells forming invadopodia were maintained under hypoxia conditions. We incubated phenotype-microarray (PM) plates containing the cells in the hypoxia chamber for 48 h in order to allow the cells to consume the residual nutrient provided in the media. Following that, cells were treated with 0.5 mM DMOG; at that time, the cells started to utilize the chemical substrates provided in each well. Interestingly, results showed that several substrates were utilized in hypoxic MDA-MB-231, specifically in the PM-M1 plate that contained primary carbohydrate and carboxylate substrates as a single nutrient source for their survival by turning purple as energy . Furtherd-maltose, d-glucose-6-phosphate, a-d-glucose-1-phosphate, d-glucose, d-mannose, turanose, d-tagatose, d-fructose-6-phosphate, and lactulose . These results were validated and confirmed by the expression of VEGF as a positive control for HIF-1α. VEGF is not only a positive control for HIF-1α, but also a well-known marker for angiogenesis and tumor growth [Moreover, the expression level of HIF-1α significantly increased under hypoxia conditions using a hypoxia chamber . Results provided evidence for using dextrin as a drug carrier to enhance the effectiveness of cancer therapy against hypoxia and cancer invasion; this could be a novel factor to focus on in hypoxia studies. A similar report used dextrin as a drug carrier in nanoparticle applications to attack a specific target in cancer therapeutics in normal conditions [On the other hand, the mentioned significant chemical substrates, such as dextrin, can potentially be a drug carrier targeting invadopodium formation under hypoxia. Our findings showed that dextrin was significantly more metabolized in hypoxia than in normoxia. Additionally, the morphology of MDA-MB-231 cells survived after 72 h incubation in hypoxia. However, the nditions . Dextrinnditions . In therThus, these findings suggest the further exploration and testing of dextrin or even other significant substrates to use them as a drug carrier to enhance the effectiveness of chemotherapy. This may improve the selectivity of the drugs to target the hypoxia region, cancer invasion, and metastasis. Taken together, our investigation into what invadopodia fuel is in a hypoxia background is still ongoing, and what we found are preliminary findings on invasion energy. Those chemical substrates as drug carriers are potential candidates to enhance the effectiveness of chemotherapy.2 and 5% CO2 incubator at 37 °C for normal conditions. For hypoxia conditions, cells were incubated for a specific amount of time in a hypoxia chamber  with 1% O2, 5% CO2, and balanced with N2, and then treated with DMOG (purchased from Sigma) at a final concentration of 0.5 mM to mimic hypoxia. Cells with low passage numbers were used in all experiments in order to get accurate results.MDA-MB-231 breast-cancer cell line was obtained from American Type Culture Collection . Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), high-glucose medium purchased from Gibco  containing FBS 10% and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere of 95% O® 488 Conjugate gelatin from pigskin  was used to coat glass coverslips prior to seeding cells. The protocol was carried out according to Enderling et al. (2008) with slight modification [2O and transferred to slides containing a drop of antifade, a mounting solution. For invadopodium quantification, 100 cells were randomly selected from each coverslip under a fluorescence microscope ; the mean percentage of cells forming invadopodia was calculated. ImageJ was used to measure the area of matrix degradation per cell of invadopodium-forming cells in different conditions according to a previous report [To coat the glass coverslip, 12 mm round coverslips were cleaned using alcohol for 90 min at 70% alcohol, and 30 min at 96% alcohol. Then, coverslips were placed on a filter paper to dry in a biosafety cabinet. After that, Oregon Greenfication . Coversls report .2 and/or treated with 0.5 mM DMOG for the indicated time. The cell lysate was done quickly to save HIF-1α from degradation by adding 200 μL of RIPA buffer mixed with protease inhibitor (100:1) on the ice plate. Following that, a PCA kit  was used to determine the concentration of the protein. Each sample contained 20 μg of total protein, which was then loaded and separated by 10% SDS-PAGE and blotted on a PVDF membrane. To block the membranes, 5% of BSA with tris buffered saline–Tween 20 (TBS-T) was added for one hour in a shaker, and then incubated overnight at 4 °C on the shaker with the following primary antibodies and dilutions: HIF-1α , MMP-2, VEGF, Cool/β-PIX , and β-actin ; antibodies were diluted with TBS-T buffer containing 5% BSA. Following that, the primary antibody was discarded and washed 3 times with TBS-T each time for 10 min. Membranes were then incubated with HRP-conjugated secondary antibodies for one hour (1:10000 for β-actin and 1:1000 for other antibodies). Then, PVDF membranes were washed with TBS-T three times, each time for 10 min, and viewed under chemiluminescence using a gel-documentation system  after incubating membranes with enhanced chemiluminescent reagents (ECL). Band quantification was measured using ImageJ software.Whole protein was extracted from MDA-MB-231 cells. Cells were grown in T25 flasks; when confluency reached 80%, cells were exposed to a hypoxia chamber of 1% Od-PBS, 2 mL of trypsin was added into each flask. Then, the cell suspension was collected and once washed with d-PBS using 4 °C centrifugation. After that, d-PBS was discarded, and 3 mL of IF-M1 medium containing 1% penicillin and streptomycin, 0.3 mM glutamine, and 5% FBS was added to the pellet. The pellet was then resuspended by pipetting up and down several times. Cell viability was ~97% using trypan blue in all experiments. Cells were seeded into PM plates; each well contained 2 × 104 cells/well in 50 μL of the medium. The number of cells and incubation time were optimized prior to the actual experiment to ensure that the cells utilized residual nutrients provided with the IF-MI medium before they adapted and switched to the saturating single-chemical substrates precoated in each well of the PM plates. Following that, normoxia plates were incubated in a humidified atmosphere of 95% air and 5% CO2 in the incubator for 48 h; then, hypoxia-group and negative-control plates were incubated for 48 h in a hypoxia chamber and placed into 37 °C. The hypoxia chamber was refilled with mixed gas every 12 h to keep cells under the hypoxia condition. The negative-control group contained only Biolog IF-M1 medium. After incubation for 48 h, 10 μL of Biolog Redox Dye Mix (MA dye) was added into each well of the normoxia group. Then, 10 μL of MA dye mixed with DMOG was added. The total concentration of DMOG in each well was 0.5 mM in the hypoxia and negative-control groups. After that, all PM plates were sealed with adhesive film and placed in the OmniLog incubator reader set at 37 °C using standard operating conditions, which collected data every 15 min over a 24 h period. Tetrazolium reduction was kinetically measured by the OmniLog system in which the kinetic readout of color formation was obtained, and tetrazolium-reduction rate was determined.The PM principle is a cell-based assay used to determine around 1400 phenotypic metabolisms of mammalian surviving cells. PM provides 96-well microplates precoated with various types of carbon and nitrogen sources in the bottoms, including fatty acids, carboxylic acids, amino acids, bi-amino acids, fatty acids, ketones, carbohydrates/starches, and alcohols . Furthert-test with a p value < 0.05. With regards to phenotype microarrays, data were analyzed using a special technique for visualization and analysis. High-throughput phenotypic data were extracted as raw kinetic profiles using OM_FM software . Then, data were inserted into the OPM package in R software as a CSV-file type for efficient and meaningful statistical analysis and visualization. Analysis steps were carried out using the OPM R package and divided into three stages: grouping, normalization, and affect identification [All performed experiments were analyzed using GraphPad Prism , and expressed as mean values with SD of three separate experiments unless otherwise indicated. Statistical significance was calculated by one-way ANOVA or Student′s fication ,45. Resud-malic acid.In this study, the carbon sources that were greedily consumed under hypoxia hold great potential for developing targeted therapy specifically for cancer cells. In this study, we found 11 chemical substrates that could be considered as fuel for hypoxia-induced invadopodium formation in MDA-MB-231 cells. One potential candidate is dextrin, a substrate that may drive invadopodia under hypoxia conditions. The findings propose an opportunity to use some types of carbon sources, like dextrin, as drug carriers to enhance the effectiveness of chemotherapy in combatting tumor hypoxia and cancer-cell invasion. Apart from that, other chemical substrates can be used as inhibitors of invadopodium formation, such as a low concentration of


Proximal femoral osteolysis was seen in 23% of hips, and MRI demonstrated ARMD in 27 of the 66 investigated hips (41%). Mean cobalt, chromium, and titanium ion concentrations were 0.82 µg/L (range 0.22–4.45), 1.51 µg/L (0.04–22.69), and 2.68 µg/L (0.26–19.56) in patients with unilateral THA, and 2.59 µg/L (0.43–24.75), 2.50 µg/L (0.26–16.75), and 3.76 µg/L (0.67–19.77), respectively in patients with bilateral THA. Twenty-nine percent of patients showed cobalt or chromium ion levels > 2 µg/L. (4) Conclusions: Despite good clinical long-term results, increased blood metal ion levels  were found in approximately one-third of asymptomatic patients, and proximal femoral osteolysis and ARMD were frequently seen in this cohort. Blood metal ion analysis appears helpful in the long-term follow-up of these patients in order to identify individuals at risk. In accordance with contemporary consensus statements, symptomatic patients with elevated metal ion levels and/or progressive osteolysis should be considered for additional CT or MARS MRI to determine the extent of soft tissue affection prior to revision surgery. Further studies are necessary to investigate the clinical relevance of ARMD in asymptomatic patients with small-head, MoM THA. Second-generation, small-head, metal-on-metal (MoM) total hip replacements were reintroduced in 1988 by Weber . High eaThe objective of the present study was (i) to evaluate the clinical and radiological results of small-head, MoM THA at long-term follow-up, (ii) to determine blood metal ion concentrations in a large cohort of patients at a minimum follow-up of 10 years, and (iii) to investigate potential risk factors associated with elevated blood metal ion levels in these patients.In this cross-sectional study, we retrospectively evaluated a consecutive series of 262 patients (284 hips) following cementless THA with a 28-mm Metasul metal-on-metal articulation. The study was approved by the ethics committee of the Heidelberg school of medicine (No. S-365/2013), and informed written consent was obtained prior to inclusion of each patient. Surgery was performed consecutively between April 1995 and November 2001 at Heidelberg University Hospital using either a modified Watson-Jones or a transgluteal lateral approach. The indication for the use of a MoM bearing at that time was young patient age and a high expected physical activity level. The mean age of patients at time of surgery was 52 years (range 21 to 74 years). At a mean follow-up of 14.5 years, 44 patients  had died and 14 (5%) were lost to follow-up, leaving 193 patients (211 hips) who were available for review . Up to tA 28-mm Metasul  MoM articulation was used in all hips. The acetabular component of this implant consists of a forged, high-carbide (0.2–0.25%) cobalt-chromium alloy liner, which is embedded in a polyethylene insert. It was used in combination with a cementless, press-fit titanium acetabular shell; 95 hips received an Allofit acetabular cup  and 58 received a Fitmore acetabular component . An uncemented straight-tapered titanium stem with a standard 12/14 mm Euro taper was used in all hips for femoral reconstruction; 128 hips received a CLS Spotorno stem  and 25 received a G2 stem .®, Voyant Health, Columbia, SC, USA), taking the inter-teardrop line as a fixed landmark [Clinical examination was performed using the Harris Hip Score. Standard pelvis anteroposterior and lateral radiographs of the hip were evaluated with regard to radiolucent lines and osteolysis. We defined periprosthetic osteolysis as a lucent zone absent of trabecular bone, which was not visible on the immediate postoperative radiograph . Radiolulandmark . In addilandmark . Blood samples were taken using a blood collection system specific for trace metal ion analysis . The first 5ml of blood were discarded and blood samples were stored at −20 °C. Whole blood metal ion analysis was performed at the Geochemical Laboratories at Heidelberg University using high-resolution, inductively-coupled, plasma-mass spectrometry . ICP-MS is currently considered one of the preferred techniques for blood metal ion measurement . All sam® for Windows®  and Graphpad Prism® . Data were evaluated descriptively as arithmetic mean, standard deviation, median, minimum, and maximum. Demographic data and mean metal ion levels were compared between the bilateral and the unilateral group using the student’s t-test. For comparison of categorical variables between the two groups, the chi-square test was used. Kaplan-Meier survivorship analysis was performed with revision for any reason as the endpoint. In the unilateral group, correlation analysis was performed using Spearman correlation coefficient and multivariate linear regression analysis in order to investigate the correlation between blood metal ion concentration and potential risk factors associated with elevated cobalt ion levels, which were defined as gender, cup inclination angle, body mass index, and follow-up length. Additionally, the relationship between periprosthetic osteolysis and blood metal ion concentrations of cobalt, chromium, and titanium was assessed using logistic regression analysis. Correlation was defined as poor (0.00 to 0.20), fair (0.21 to 0.40), moderate (0.41 to 0.60), good (0.61 to 0.80), or excellent (0.81 to 1.00). All tests were two-sided and a p-value < 0.05 was considered significant.Statistical analysis was performed using the software SPSSThe cumulative survival rate at 10 years, using revision for any reason as the endpoint, was 96% ; 92–98%; 235 hips at risk) and 94%  at a mean follow-up of 14 years . Of the The mean Harris Hip Score of the cohort was 90 points (range 40 to 100) at the time of follow-up. The mean inclination angle of the acetabular component was 42 degrees (range 29–50 degrees). No femoral component showed radiographic signs of loosening. Radiographs demonstrated femoral osteolysis in 23% of the hips and radiolucent lines > 2 mm in 13% of hips. Osteolysis and radiolucent lines were predominantly located in the proximal Gruen zones. Their distribution is illustrated in p < 0.001), and fair correlation between chromium and titanium ion levels  and between body mass index and cobalt ion levels . However, in multivariate analysis, none of the tested variables was proven as a risk factor for elevated metal ion levels. Logistic regression analysis showed no association between the presence of periprosthetic osteolysis and blood metal ion levels of cobalt , chromium , or titanium .A total of 139 patients were eligible for blood metal ion analysis, with the Metasul bearing being the only known source for cobalt or chromium ion release . The demSmall-diameter, metal-on-metal implants are supposed to be at low risk of developing ARMD, and a systematic follow-up comparable to conventional THA is considered to be sufficient due to the good clinical mid- and long-term results reported in the literature . CurrentTo our knowledge, the present study represents the largest cohort of patients following small-head, MoM THA investigated with blood metal ion analysis at long term follow-up. Metal ion levels may vary significantly depending on the medium  and the technique (AAS vs. ICP-MS) used for analysis, which limits comparison among published studies . Migaud For hip resurfacing and large-head, metal-on-metal THA, different risk factors for implant failure and elevated metal ion levels could be identified, such as high cup inclination angles or female sex ,24. SidaWe found an acceptable clinical outcome for this bearing type according to the NICE recommendations , with a There are some limitations to our study. Five percent of patients were lost to follow-up and a further 10% declined to participate in blood metal ion analysis. Also, no CT-scans were carried out in order to avoid additional radiation exposure. As a consequence, the rate of osteolysis could have been underestimated, especially around the acetabular components. Furthermore, MRI was only performed in 66 of the 189 hips (35%) that were available for clinical and radiological assessment, which could have resulted in a selection bias regarding the prevalence of ARMD. The fact that only 66 of the 107 advised patients with elevated metal ion levels agreed to participate in MRI assessment was mainly attributed to long travel distances and/or the absence of symptoms . FurtherThe present study demonstrates good clinical results for cementless, 28 mm, MoM, THA at long-term follow-up, with a cumulative survival rate of 94% after 14 years. However, increased blood metal ion levels  were found in approximately one-third of asymptomatic patients, and proximal femoral osteolysis and ARMD were frequently seen in this cohort. Blood metal ion analysis appears helpful in the long-term follow-up of these patients in order to identify individuals at risk. In accordance with contemporary consensus statements , symptom